This invention relates generally to immunoassays for microbial detection, and more particularly to methods for rapidly detecting pathogenic microbes.
Rapid detection of biological agents used as warfare agents, terrorist threat agents, and emerging diseases are significant military and civilian challenges. For instance, biological agents when effectively prepared are extremely potent and could be disseminated to incapacitate or kill thousands of persons. It has been reported that during the Gulf War, U.S. and allied forces suffered from a lack of reliable biological agent detection systems. While a number of detection systems were developed to overcome this problem, several of these methods fail to reliably provide the accuracy, sensitivity, and speed desired. In addition, worldwide 22 million people die annually of infectious diseases. Bacterial infections are responsible for a high percentage of these fatal infections. Accordingly, there is a need for methods that can quantitatively and sensitively detect bacterial pathogens for multiple medical and industrial purposes, including biological warfare defenses.
A bacterial detection method should be reliable. That is, it should measure relatively the exact number of the pathogen multiple times under the same conditions. Moreover, speed is critical to the acceptability of the detection method.
Traditionally, detection of microorganisms relies on the same biochemical basis developed by Pasteur and others in the last century and the disk diffusion method developed by Kirby-Bauer for antimicrobial susceptibility testing (e.g., Wright, et al., Epidemiol. Infec. 113:31-39 (1994)). During the last decade, emphasis had been on automation of these basic biochemical tests. This has accelerated the identification process, but even with the most sophisticated system, the process still takes at least roughly thirty hours under optimal conditions for a precise diagnosis of bacterial infection.
An example of a bacterial pathogen is Escherichia coli BL21 Star (DE3) pLysS, which is a specific strain of E. coli that can potentially causes moderate to severe diseases for organism's eyes, skin, lung and digestive tract (Invitrogen Corporation, MSDS for BL21 Star™ (DE3) One Shot™ Chemically Competent E. coli.). Clinical symptoms include eye tearing, reddening, and temporary vision impairment (cloudy or blurred vision); skin irritation, defatting, and dermatitis; dizziness, weakness, fatigue, nausea, and headache if inhaled; abdominal discomfort, nausea, vomiting and diarrhea if ingested. Magnetic immunoassay for isolation E. coli was reported by Wright et al., Epidemiol Infec. 113:31-39 (1994) with an assay time of approximately 24 hrs. Detection methods for E. coli based on enzyme-linked immunosorbent assays (ELISA) (Padhye & Doyle, J. Clin. Microbiol. 29:99-103 (1991)) and polymerase chain reaction (PCR) (Johnson, et al. Appl. Environ. Microbiol. 64:4390-95 (1998)) have been developed and improvements in their performance time and sensitivity are ongoing. PCR detection methods have the possibility of single cell detection with the potential of taking less than eight hours to perform (Ogunjimi & Choudray, FEMS Immunol. Med. Microbiol. 23:213-20 (1991)). However, some molecular methods of bacterial detection have not been fully accepted by routine microbiology testing laboratories. This may be due to the need for relatively expensive equipment and associated specialist skills to perform the analyses (Bayliss, MAFF Research Program FS 12, Detection and Separation of Pathogens and their Toxins. MAFF UK, Center for Applied Microbiology and Research, Portion Down (1999)). By comparison, immunological detection tests for bacterial pathogens (including latex agglutination, immunomagnetic separation, lateral flow immunoassays and ELISA) are used frequently. Furthermore, ELISA detection methods have sensitivities of 10−5-107 bacterial cells/ml−1 (Kim, et al., J. Sci. Food Agric. 79:1512-18 (1999)) and require overnight enrichment of the sample prior to analysis (Feldsine, Food Biol. Contam. 80:517-29 (1997)). Some sensitive immunological methods have been developed using electro-chemiluminescence (Yu & Bruno, Appl. Environ. Microbiol. 62:587-92 (1996) and rapid flow through systems (Abdelhamid et al., Biosensors Bioelect. 14:309-16 (1999)). Current detection methods have led to the possibility of detection within a single working day.
Hand held devices for rapid detection of pathogens have been proposed to be used by emergency medical services, fire and rescue services, hazmat teams, and other first responders. However, pathogen detection kits currently available are not accurate for on-the-scene decision-making and require significant user training.
In addition, the possibility of relatively low concentrations of collectable bacteria at a particular site of investigation/testing would be expected to challenge or refute detection by traditional culture methods. For instance, studies suggest that only 1-10% of ambient bacteria are culturable (Padhye & Doyle, J. Clin. Microbiol. 29:99-103 (1991)). According to the Center for Biological Defense, aerosolized bacteria are of major concern when used for warfare or terrorist attack. It would therefore be desirable to provide a portable, lightweight, and easy-to-use device for pathogen detection. It would also be desirable to provide means for rapidly detecting pathogens with high sensitivity.